Uncharacterized Bacterial StructuresRevealed by Electron Cryotomography

Megan J. Dobro,a Catherine M. Oikonomou,b Aidan Piper,a John Cohen,a

Kylie Guo,b Taylor Jensen,b Jahan Tadayon,b Joseph Donermeyer,b Yeram Park,b

Benjamin A. Solis,c Andreas Kjær,d Andrew I. Jewett,b Alasdair W. McDowall,b

Songye Chen,b Yi-Wei Chang,b Jian Shi,e Poorna Subramanian,b Cristina V. Iancu,f

Zhuo Li,g Ariane Briegel,h Elitza I. Tocheva,i Martin Pilhofer,j Grant J. Jensenb,k

Hampshire College, Amherst, Massachusetts, USAa; California Institute of Technology, Pasadena, California,USAb; University at Albany, SUNY, Albany, New York, USAc; University of Southern Denmark, Odense,Denmarkd; National University of Singapore, Singapore, Republic of Singaporee; Rosalind Franklin University ofMedicine and Science, Chicago, Illinois, USAf; City of Hope, Duarte, California, USAg; Leiden University, SylviusLaboratories, Leiden, Netherlandsh; University of Montreal, Montreal, Quebec, Canadai; ETH Zurich, Zurich,Switzerlandj; Howard Hughes Medical Institute, Pasadena, California, USAk

ABSTRACT Electron cryotomography (ECT) can reveal the native structure and arrange-ment of macromolecular complexes inside intact cells. This technique has greatly ad-vanced our understanding of the ultrastructure of bacterial cells. We now view bacteriaas structurally complex assemblies of macromolecular machines rather than as undiffer-entiated bags of enzymes. To date, our group has applied ECT to nearly 90 differentbacterial species, collecting more than 15,000 cryotomograms. In addition to knownstructures, we have observed, to our knowledge, several uncharacterized features inthese tomograms. Some are completely novel structures; others expand the featuresor species range of known structure types. Here, we present a survey of these un-characterized bacterial structures in the hopes of accelerating their identification andstudy, and furthering our understanding of the structural complexity of bacterialcells.

IMPORTANCE Bacteria are more structurally complex than is commonly appreciated.Here we present a survey of previously uncharacterized structures that we observedin bacterial cells by electron cryotomography, structures that will initiate new linesof research investigating their identities and roles.

KEYWORDS bacteria, electron cryotomography, bacterial ultrastructure,uncharacterized structures, electron microscopy

The history of cell biology has been punctuated by advances in imaging technology.In particular, the development of electron microscopy (EM) in the 1930s produced

a wealth of new information about the ultrastructure of cells (1). For the first time, thestructure of cell envelopes, internal organelles, cytoskeletal filaments, and even largemacromolecular complexes like ribosomes became visible. A further advance came inthe 1980s and 1990s with the development of electron cryotomography (ECT) (2),which allows small cells to be imaged intact in 3D in a near-native, “frozen-hydrated”state to “macromolecular” (�5 nm) resolution, without the limitations and artifacts ofmore traditional specimen-preparation methods (3).

ECT has helped reveal the previously unappreciated complexity of “simple” bacterialcells. Our group has been using ECT to study bacteria for more than a decade,generating more than 15,000 tomograms of 88 different species. These tomogramshave revealed new insights into, among other things, the bacterial cytoskeleton (4–7),cell wall architecture (8, 9), morphogenesis (10), metabolism (11), motility (12–15),

Received 10 March 2017 Accepted 27 May2017

Accepted manuscript posted online 12June 2017

Citation Dobro MJ, Oikonomou CM, Piper A,Cohen J, Guo K, Jensen T, Tadayon J,Donermeyer J, Park Y, Solis BA, Kjær A, JewettAI, McDowall AW, Chen S, Chang Y-W, Shi J,Subramanian P, Iancu CV, Li Z, Briegel A,Tocheva EI, Pilhofer M, Jensen GJ. 2017.Uncharacterized bacterial structures revealedby electron cryotomography. J Bacteriol199:e00100-17. https://doi.org/10.1128/JB.00100-17.

Editor Thomas J. Silhavy, Princeton University

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Grant J. Jensen,jensen@caltech.edu.

RESEARCH ARTICLE

crossm

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 1Journal of Bacteriology

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

chemotaxis (16), sporulation (17), cell-cell interactions (18), and phage infection (19) (fora summary with more references from our and others’ work, see reference 20).

A major hurdle in such studies is identifying the novel structures observed intomograms. In some cases, we have identified structures by perturbing the abundance(either by knockout or overexpression) of candidate proteins (21). In others, we haveused correlated light and electron microscopy (CLEM) to locate tagged proteins ofinterest (22, 23). In one striking example, we observed 12- and 15-nm-wide tubes in ourtomograms of Vibrio cholerae cells. Ultimately, in collaboration with John Mekalanos’group, we identified them as type VI secretion systems (T6SS), which immediately ledto the insight that the bacterial T6SS functions as a phage-tail-like, contractile moleculardagger (18).

Many other novel structures we have observed, though, remain unidentified. Insome cases, we have published papers describing the novel structures seen in aparticular species (e.g., references 24 and 25), but many have never been published. Wetherefore conducted a visual survey of the tomograms collected by our group, curatedin the Caltech Tomography Database (26), as of 2015 and present here a catalog ofpreviously undescribed bacterial structures. Some structures are, to our knowledge,completely novel; others belong to known types but present additional features or anexpanded species range. We hope that sharing these images will help spur theiridentification and study, contributing to our expanding understanding of bacterial cellbiology. In addition, we look forward to a future in which custom microbes aredesigned for diverse medical and industrial purposes; an expanded “parts list” ofstructures to be repurposed will aid in this effort.

RESULTS AND DISCUSSION

We performed a visual inspection of approximately 15,000 tomograms of intactfrozen-hydrated cells belonging to 88 species and identified what we believed to benovel structures. A summary of the results of this survey is shown in Table S1 in thesupplemental material, with features observed, species range, and frequency listed foreach structure type. For full tomographic (3D) views of each feature, see reference 27;to view the figures in virtual reality, see reference 75.

Extracellular structures: external appendages. Prosthecobacter debontii is a bac-terial species from the poorly studied phylum Verrucomicrobia. Each vibroid P. debontiicell possesses an appendage (prostheca), similar to the stalk of Caulobacter crescentus(28). In several tomograms of P. debontii, we observed novel extracellular appendagesalong the prosthecae, apparently attached to the cell membrane. Individual cellsdisplayed up to 30 such appendages, which exhibited consistent sizes (�20 nm wideand �50 nm long) and shapes (Fig. 1A). Subtomogram averaging of 105 particlesrevealed a distinctive structure. Extending outward from the cell membrane, five legswere attached to a disc, which in turn connected to a smaller disc and a long neckregion (Fig. 1B and C). Individual particles showed that the structure culminated in twoantenna-like filaments, which were likely lost in the average due to conformationalvariability. The appendages were observed in multiple cultures of the strain. While itremains unclear whether they originated intracellularly or extracellularly, no free-floating appendages were ever observed in the extracellular space. They may representnovel bacterial attachment organelles or appendages for nutrient acquisition, whichhas been proposed for a similar structure, the Caulobacter stalk. Under phosphate-limited conditions, Caulobacter grew elongated stalks (29). This increase in cell surfacearea with respect to cell volume was hypothesized to allow increased phosphateuptake (30). However, the presence of diffusion barriers challenges this view (31). Theappendages could also be a novel secretion system (though we might expect a cellenvelope spanning complex) or a novel bacteriophage (though there is a notable lackof a capsid-like density).

We observed a different novel extracellular appendage in tomograms of cell polesof Azospirillum brasilense, a plant growth-promoting bacterium of the Alphaproteobac-teria class with a curved-rod morphology. Thin hooks were seen extending from the cell

Dobro et al. Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 2

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

surface (Fig. 2). Individual cells exhibited dozens of hooks, each �3 nm wide and �75nm long, associated with the outer membrane. The cells are large, and most tomo-grams included only the cell pole region, so while hooks were observed only at the poleregion, they may also occur elsewhere on the cell. Hooks were seen in �90% ofwild-type cells as well as in a strain in which the operon encoding the Che1 chemotaxissystem was deleted. They were seen in �50% of cells in which the Che4 chemotaxissystem operon was deleted, and none were seen in cells lacking both the Che1 and

FIG 1 Novel Prosthecobacter debontii appendages. Multiple external appendages (arrowheads) wereobserved by ECT on P. debontii prosthecae. (A) A central tomographic slice is shown, with a singleappendage enlarged in the inset. Subtomogram averaging revealed the structure in more detail. (B) Side(top) and top (bottom) views show the characteristic disc-like densities and the five legs attaching to thecell surface. The red box shows which view was used to rotate the image 90° for the bottom image. (C)3D isosurface of the average, seen from the side and top (inset). Bars, 50 nm (A) and 20 nm (inset).

FIG 2 Novel Azospirillum brasilense hooks. Many hook-like structures were observed on the surface of A.brasilense cells. A central tomographic slice is shown, with arrowheads indicating hooks. A single hookis shown enlarged at right. Bars, 50 nm.

ECT of Novel Bacterial Structures Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 3

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

Che4 operons. A. brasilense is a well-studied plant growth-promoting bacterium. Cellsattach to plant roots through a two-step process (32), a rapid reversible adsorption,thought to be mediated by the polar flagellum, and a slow irreversible anchoring,thought to be mediated by an as-yet unidentified surface polysaccharide (33). A recentstudy reported that mutants in components of the Che4 chemotaxis system aredefective in this root colonization (34). A. brasilense cells also attach to conspecifics inthe presence of elevated oxygen levels (35). Interestingly, it has been shown thatmutants in components of the Che1 chemotaxis system form such attachments morerapidly than wild-type cells (36). The hooks we observed are vaguely reminiscent of thegrappling hook-like structures that an archaeal species uses to anchor itself in biofilms(37), though those hooks were longer fibers with barbs. Those archaeal cells demon-strated very strong adhesion to a variety of surfaces as well as to each other. It istempting, therefore, to speculate that the hooks shown here play a similar role inadhesion, to either other A. brasilense cells or plant roots.

In cells of strain JT5 (a rod-shaped bacterium isolated from termite gut and relatedto the Dysgonomonas genus), we observed abundant fimbriae concentrated at the cellpoles (Fig. 3). Fimbriae were also observed at the cell body but were much moreconcentrated at the cell poles. They were present in cells grown on cellulose or xylan,as well as under a starvation-inducing condition. Their width (�4 nm), apparentflexibility, density on the cell envelope, and inhomogeneous distribution around thecell is consistent with curli, functional amyloids secreted by the type VIII secretionsystem that are involved in adhesion (38, 39). Curli systems are relatively divergent atthe sequence level but are remarkably widespread phylogenetically, and the geneswere reported to be present in Bacteroidetes (the phylum containing Dysgonomonas)(40). The appendages we observed in strain JT5 may therefore play a role in adhesionin the environment of the termite gut.

FIG 3 Strain JT5 fimbriae. Examples are shown from two cells of strain JT5 (related to the Dysgonomonasgenus) exhibiting abundant fimbrae at the cell pole. (A) Central slice revealing overall cell morphology;(B to D) slices at progressive z-heights through a cryotomogram of a cell of strain JT5 (related to theDysgonomonas genus). Abundant fimbriae can be seen at the cell pole. Bars, 100 nm.

Dobro et al. Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 4

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

Intracellular structures. (i) Nanospheres. In two Vibrio cholerae cells (one from aC6706 lacZ mutant [41] and one from a ΔctxA ΔtcpB strain [15]) we observed clustersof nanospheres, hollow granules with thick walls (Fig. 4). The diameter of the nano-spheres ranged from �18 to 37 nm, and the walls were �4 to 10 nm thick. They werepleomorphic; most were roughly spherical, but some were oblong or comma shaped.Each cluster contained about two dozen nanospheres. The clusters were observed atthe cell periphery, near the inner membrane (although the clusters were large enoughto extend to the center of the cell), and were always observed near a filament arraystructure (discussed below).

(ii) Filaments, bundles, arrays, chains and meshes. One of the strengths of ECTimaging is its power to resolve cytoskeletal elements in small bacterial cells. In additionto those we have already identified, we observed many novel filamentous structures intomograms, including filament arrays, bundles, chains, and meshes (Fig. 5). In Hy-phomonas neptunium, we observed long helical filament bundles in the prosthecae that

FIG 4 Novel Vibrio cholerae nanospheres. Clusters of nanospheres were observed in two cryotomogramsof V. cholerae cells (central slices shown in panels A and C). “N” indicates nanospheres; “A” indicatesassociated filament array. (B) Segmentation of the cluster seen in panel A, with outer and innermembranes in magenta and cyan, respectively, and nanospheres in green. A clipping plane cuts throughthe 3D segmentation revealing the thick walls and hollow centers of the nanospheres. Bars, 100 nm.

ECT of Novel Bacterial Structures Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 5

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

connect dividing cells (Fig. 5A). The helix width was 9.5 � 1.5 nm, the spacing betweencross-densities 6.0 � 0.3 nm, and the helical pitch was �26°. H. neptunium divides byasymmetric budding (42), and the genome of the parent cell is passed to the daughtercell through the narrow prostheca connecting the two cells (43). We observed that thehelical structure was straightened in cells treated with ethidium bromide (an interca-lator known to unwind DNA [44]) (Fig. 5B). We therefore propose that the helix iscomposed of supercoiled DNA, with each visible filament a DNA duplex connected toadjacent duplexes by cross-densities formed by an unidentified protein.

In Helicobacter pylori cells, we observed extensive filament bundles. In one cell in anearly stage of lysis, such bundles were observed throughout most of the cell (Fig. 5C).In V. cholerae we observed filament arrays resembling a honeycombed mesh (Fig. 5D).

FIG 5 Filament bundles, arrays, and chains. Hyphomonas neptunium division stalks contained helical bundles (A) thatstraightened when cells were treated with ethidium bromide (B). The right side of panel A shows a 3D segmentation of thehelical bundle, with side and top views of subtomogram averaged insets. Labeled dimensions are in nanometers. (C) Largefilament bundles in Helicobacter pylori. (D) A long mesh-like filament array in Vibrio cholerae, with segmentation at right. (E)A more typical V. cholerae filament array. (F to J) Filament arrays in Thiomonas intermedia (F), Hyphomonas neptunium (G),Hylemonella gracilis (H), Halothiobacillus neapolitanus c2 (I), and Mycobacterium smegmatis (J). (K) A chain in Prosthecobactervanneervenii. (L to M) Filament arrays in Prosthecobacter debontii. (N) A filament array in a starved Campylobacter jejuni cell.Bars, 100 nm (A, B, D to J, L to N) and 50 nm (C and K).

Dobro et al. Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 6

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

These arrays varied in length but were usually fairly short (�100 nm in length andwidth), like the example shown in Fig. 5E. This is the structure we observed near thenanosphere clusters. Filament arrays exhibited different morphologies in otherspecies. Thiomonas intermedia cells contained untwisted arrays that were �48 nmthick and �30 nm wide (Fig. 5F). In addition to the prosthecal helix describedabove, H. neptunium cells also contained a bundle of twisting filaments laddered bycross-densities (Fig. 5G). These bundles were �40 nm thick and �75 nm wide. In aHylemonella gracilis cell we observed a helical bundle of filaments that varied inwidth and could be related to the nucleoid (Fig. 5H). In Halothiobacillus neapolita-nus c2 cells grown in limited CO2 for several hours, we observed linear filamentarrays with prominent cross-densities spaced 7 � 0.8 nm apart (Fig. 5I). Mycobac-terium smegmatis displayed straight arrays �80 nm thick and wide, comprisingsegments of pitched filaments (Fig. 5J). Filament arrays were also seen in multiplespecies of Prosthecobacter; P. vanneervenii contained linear chains (Fig. 5K), and oneP. debontii cell contained a straight array similar to those observed in T. intermedia(Fig. 5L), as well as mesh-like arrays spanning the width of the prostheca (Fig. 5M).

In starving Campylobacter jejuni cells, we observed regular filament arrays (Fig. 5N).When subjected to environmental or cellular stress, some bacteria, including Escherichiacoli, have been shown to reorganize their DNA into protective crystalline arrays (45).Since then, additional nucleoid associated proteins have been identified that organizeDNA into higher order structures in stationary-phase or stress conditions (46, 47). Thestructures we observed in C. jejuni resemble those seen in E. coli cells overexpressingthe protective DNA binding protein Dps (45) and may, therefore, represent such anucleoprotein array.

Several other proteins have been shown to copolymerize with DNA into filamentsfor various functions, including RecA (homologous recombination) (48) and MuB frombacteriophage Mu (DNA transposition) (49). The width of such filaments in vitro (�10nm) is similar to widths we observed in cells; it is possible that some of the structuresin Fig. 5 represent these DNA-related processes. Other bacterial proteins form filamentsto regulate their function, and it has been suggested that this property may have beencoopted in the evolution of the cytoskeleton (50). We previously observed suchfilaments of CTP synthase in tomograms of C. crescentus cells (21). Another protein,alcohol dehydrogenase, forms plaited filaments �10 nm wide, called spirosomes, inmany bacteria capable of anaerobic metabolism (51, 52). It is possible that some of thefilament arrays and chains that we observed in tomograms were filaments formed bythese or other, yet uncharacterized, proteins.

In addition to filament arrays and bundles, we observed individual or pairedfilaments in nearly every species imaged. Examples are shown in Fig. 6. (Note that dueto their ubiquity, statistics are not included in Table S1 in the supplemental material.)Filaments were seen with various orientations in the cytoplasm (Fig. 6A to C), as wellas running alongside the membrane (Fig. 6D and E). Consistent with our previous work(53), we did not observe any filaments immediately adjacent to the membrane aspredicted by some studies of MreB (e.g., references 54 and 55). (Note that we didobserve filaments corresponding to the known types of MamK [4, 56], FtsZ [5, 57], andbactofilins [58] but we do not show them here since they have already been charac-terized.) Paired filaments have been shown to function in plasmid segregation, so it ispossible that some paired filaments that we observed were such ParM or TubZstructures (59, 60).

(iii) Tubes. In addition to the known types of tubes we reported earlier, such asbacterial microtubules (6) and type VI secretion systems (18), we observed several noveltubular structures in bacterial cells (Fig. 7). In Thiomicrospira crunogena we found largetubes (18.6 � 1.8 nm in diameter) containing eight outer protofilaments surroundinga central protofilament (Fig. 7A). H. neapolitanus c2 cells also contained large tubes(16.7 � 0.7 nm in diameter) with a central filament (Fig. 7B). In several other species, weobserved hollow tubes of varying dimensions: 8.9 � 0.3 nm in diameter in Bdellovibrio

ECT of Novel Bacterial Structures Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 7

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

bacteriovorus (Fig. 7C), 14.3 � 1.7 nm in T. intermedia (Fig. 7D), and 8.3 � 0.5 nm in H.neptunium (Fig. 7E). H. neptunium cells also contained many rings of similar diameter.In fact, we observed rings in many species, which could be assembly or disassemblyintermediates of tubes.

In addition to isolated rings, in one case we observed an organized array of rings.One slightly lysed (a condition that flattens the cell and increases image quality) H.pylori cell contained a striking array of about two dozen evenly spaced rings near thecytoplasmic membrane (Fig. 7F). Each ring was �6 nm in diameter and �20 nm(center-to-center distance) from its neighbors in the square lattice.

FIG 6 Single and paired filaments. Tomographic slices showing paired filaments in Campylobacter jejuni (A) andThiomicrospira crunogena (B) and membrane-aligned filaments in Shewanella putrefaciens (C), Prosthecobacterdebontii (D), and Prosthecobacter fluviatilis (E) (the arrow shows a filament just under the inner membrane). Bars,100 nm (A to D) and 50 nm (E).

FIG 7 Tubes and rings. Tubes observed in Thiomicrospira crunogena (A), Halothiobacillus neapolitanus c2 (B),Bdellovibrio bacteriovorus (C), Thiomonas intermedia (D), and Hyphomonas neptunium (E). In each panel, tomo-graphic slices show a side view (above) and a top view (bottom). (F) An array of rings observed in Helicobacter pylori.Bars, 10 nm (A, C, and E), 20 nm (B and D), and 100 nm (F).

Dobro et al. Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 8

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

(iv) Vesicles. In contrast to eukaryotic cells, relatively little is known about membraneremodeling in bacteria. Compartmentalized cells in the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum have been shown to contain homologs of eukaryoticmembrane trafficking proteins (61) and exhibit endocytosis-like protein uptake (62). Anadditional potential membrane-remodeling system based on FtsZ homologues is morewidespread across bacteria, but its function remains unknown (63).

Despite this limited evidence for membrane remodeling in bacteria, we observedintracellular vesicles in nearly every species imaged. They exhibited various sizes,shapes, membrane layers, and contents and were frequently found near the cytoplas-mic membrane. Figure 8 shows examples of round and horseshoe-shaped vesicles.Round vesicles were found in nearly every species imaged; therefore, no statistics forthem are compiled in Table S1 in the supplemental material. Most round vesicles wereempty (density similar to background [e.g., Fig. 8A to C]). One of these vesicles,observed in a lysed cell (improving clarity by reducing cytoplasmic crowding), exhibitedregularly spaced protein densities around its exterior (Fig. 8C). Others were at leastpartially filled with denser material (e.g., Fig. 8D to F). In two Myxococcus xanthus cellsoverexpressing a fluorescent fusion of a periplasmic protein (PilP-sfGFP), we observedround vesicles containing a dense amorphous core (Fig. 8F). These could be a novelform of membrane-bound inclusion bodies, perhaps packaged from the periplasm. Ineight species, we observed horseshoe-shaped vesicles (Fig. 8G and H).

FIG 8 Round and horseshoe-shaped vesicles. Tomographic slices showing examples of round vesicles in Escherichiacoli (A) (segmentation shown at right), Helicobacter pylori (B), Helicobacter hepaticus (C), Myxococcus xanthus (D),Caulobacter crescentus (E), and Myxococcus xanthus overexpressing PilP-sfGFP (F). Examples of horseshoe-shapedvesicles in Ralstonia eutropha (G) and Prosthecobacter fluviatilis (H), with 3D segmentations shown at right. In thesegmentation in panel A, outer and inner membranes are in magenta and cyan, respectively, and vesicles are ingreen. Bars, 50 nm.

ECT of Novel Bacterial Structures Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 9

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

Flattened vesicles (Fig. 9A to F) were less common than round vesicles, and wereusually observed near membranes or wrapping around storage granules (Fig. 9E),suggesting a possible functional relationship. Flattened vesicles were usually empty.One T. intermedia cell contained a stack of flattened vesicles (Fig. 9A). Flattened vesicleswere particularly prevalent in C. crescentus cells (Fig. 9B to E). P. debontii cells containedflattened vesicles that neither ran along the membrane nor wrapped around granules(Fig. 9F). Since the lowest energy shape of a liposome is a sphere, it is likely that thevesicles were flattened by cytoplasmic pressure or some other constraint such as anassociated protein.

Many cells contained nested vesicles, with diverse sizes and shapes, as well assubcellular locations (Fig. 9G to L). In some nested vesicles, densities were observedbridging the inner and outer membranes (Fig. 8A and 9G and H). Cells of strain JT5exhibited multiple nested vesicles of uniform shape and size (Fig. 9L).

We also observed periplasmic vesicles in many species (Fig. 10). They were typicallyempty and exhibited great variability in size, shape, and abundance. In some cases, theywere even seen to form branching networks (Fig. 10A). As with cytoplasmic vesicles,

FIG 9 Flattened and nested vesicles. Examples of flattened vesicles in Thiomonas intermedia (A), Caulobactercrescentus (B to E), and Prosthecobacter debontii (F). Note storage granules in panels E and F, shown in orange inthe segmentation panel E (G to L). Examples of nested vesicles in Serpens flexibilis (G), Caulobacter crescentus (H),Borrelia burgdorferi (I), Vibrio cholerae (J), Caulobacter crescentus with segmentation (K), and strain JT5 (L). (L, inset)Shows an enlargement of central vesicle, and a 3D segmentation of the visible portion of the cell is shown below.In segmentations, outer and inner membranes are shown in magenta and cyan, respectively, and vesicles are ingreen. Bars, 50 nm.

Dobro et al. Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 10

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

they were most abundant in cells showing signs of stress (rupture of inner or outermembrane, separation of inner and outer membrane, lysis, or membrane blebbing).

Conclusions. Here we present the results of a survey of, to our knowledge, unchar-acterized bacterial structures that we have observed in our work over the past 10 ormore years. We hope that further study will identify them and their functions. Already,they signal the wealth of complexity still to be discovered in bacterial cells.

MATERIALS AND METHODSStrains and growth. Unless otherwise noted, bacterial strains were wild type and grown in

species-standard medium and conditions to mid-log or early stationary phase. Azospirillum brasilensecultures were switched to nitrogen-free medium for �16 h prior to imaging to induce nitrogen fixationand digestion of storage granules that decrease image quality. Predatory Bdellovibrio bacteriovorus cellswere cocultured with Vibrio cholerae strain MKW1383. Helicobacter pylori cells were cultured with humangastric carcinoma cells. Vibrio cholerae and Borrelia burgdorferi were grown according to conditions inreferences 64 and 65). E. coli were grown according to conditions in references 16 and 66). Caulobacter

FIG 10 Periplasmic vesicles. Examples of periplasmic vesicles in Caulobacter crescentus (A), Helicobacter pylori (�),Brucella abortus (C), Thiomonas intermedia (D), Hyphomonas neptunium (E), Myxococcus xanthus (F), and Halothio-bacillus neapolitanus c2 (G). In each panel, a central tomographic slice is shown, as well as a segmentation withouter and inner membranes in magenta and cyan, respectively, and vesicles are in green. Bars, 50 nm (A to F) and100 nm (G).

ECT of Novel Bacterial Structures Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 11

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

crescentus were grown according to conditions in reference 67. Prosthecobacters were all grownaccording to conditions in reference 6. Hyphomonas neptunium was grown according to conditions inreference 68.

In all cases, samples of cells in growth medium were mixed with BSA-treated 10 nm colloidal goldfiducials (Sigma), applied to glow-discharged EM grids (Quantifoil), and plunge-frozen in a liquidethane-propane mixture (69). Grids were maintained at liquid nitrogen temperature throughout storage,transfer, and imaging.

Electron cryotomography. The references to growing conditions above also provide specific datacollection settings. Generally, plunge-frozen samples were imaged using either a Polara or Titan Krios 300kV FEG transmission electron microscope (FEI Company) equipped with an energy filter (Gatan). Imageswere recorded using either a lens-coupled 4,000 by 4,000 UltraCam CCD (Gatan) or a K2 Summit directelectron detector (Gatan). Tilt-series were recorded from �60° to �60° in 1 to 2° increments, with defociof �6 to 12 �m and a cumulative dose of �100 to 200 e�/Å2. Tilt-series were acquired automaticallyusing either Leginon (70) or UCSF Tomography (71) software. Tomographic reconstructions werecalculated using either the IMOD software package (72) or Raptor (73). 3D segmentations and movieswere produced with IMOD (72). Subtomogram averages were calculated using PEET software (74).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/JB.00100-17.

SUPPLEMENTAL FILE 1, PDF file, 0.1 MB.

ACKNOWLEDGMENTSWe thank our collaborators who provided strains for imaging: Andrew Camilli

(Streptococcus pneumoniae), Eric Matson (strain JT5), Gladys Alexandre (Azospirillumbrasilense mutants), Lotte Søgaard-Andersen, Simon Ringgaard, and Matthew K. Waldor(Vibrio cholerae wild type and mutants), Michael Marletta (Shewanella putrefaciens), andGordon Cannon and Sabine Heinhorst (Halothiobacillus neapolitanus and Thiomonasintermedia). We also thank members of the Jensen lab for their helpful discussions.

We declare no competing interests.M.J.D. and G.J.J. conceived the idea for the study; M.J.D., C.M.O., A.P., J.C., K.G., T.J.,

J.T., J.D., Y.P., A.K., A.I.J., M.P., S.C., E.I.T., Y.-W.C., A.B., J.S., Z.L., P.S., C.V.I., B.A.S., andA.W.M. performed formal analysis and investigation; M.J.D. and C.M.O. wrote andprepared the original article draft; M.J.D., C.M.O., and G.J.J. wrote, reviewed, and editedthe final article draft; M.J.D. and G.J.J. acquired funding; G.J.J. provided resources; andM.J.D. and G.J.J. supervised the study.

This work was supported by the Hampshire College Dr. Lucy fund and the Collab-orative Modeling Center, NIH grant R01 AI27401 (to G.J.J.), the Beckman Institute atCaltech, the Gordon and Betty Moore Foundation, the Human Frontier Science Pro-gram, the Howard Hughes Medical Institute, and the John Templeton Foundation aspart of the Boundaries of Life project.

The opinions expressed in this publication are those of the authors and do notnecessarily reflect the views of the John Templeton Foundation.

REFERENCES1. Ruska E. 1987. Nobel lecture. The development of the electron micro-

scope and of electron microscopy. Biosci Rep 7:607– 629.2. Koster AJ, Grimm R, Typke D, Hegerl R, Stoschek A, Walz J, Baumeister W.

1997. Perspectives of molecular and cellular electron tomography. JStruct Biol 120:276 –308. https://doi.org/10.1006/jsbi.1997.3933.

3. Pilhofer M, Ladinsky MS, McDowall AW, Jensen GJ. 2010. Bacterial TEM:new insights from cryo-microscopy. Methods Cell Biol 96:21– 45. https://doi.org/10.1016/S0091-679X(10)96002-0.

4. Komeili A, Li Z, Newman DK, Jensen GJ. 2006. Magnetosomes are cellmembrane invaginations organized by the actin-like protein MamK.Science 311:242–245. https://doi.org/10.1126/science.1123231.

5. Li Z, Trimble MJ, Brun YV, Jensen GJ. 2007. The structure of FtsZfilaments in vivo suggests a force-generating role in cell division. EMBOJ 26:4694 – 4708. https://doi.org/10.1038/sj.emboj.7601895.

6. Pilhofer M, Ladinsky MS, McDowall AW, Petroni G, Jensen GJ. 2011Microtubules in bacteria: ancient tubulins build a five-protofilamenthomolog of the eukaryotic cytoskeleton. PLoS Biol 9:e1001213. https://doi.org/10.1371/journal.pbio.1001213.

7. Swulius MT, Jensen GJ. 2012. The helical MreB cytoskeleton in Esche-richia coli MC1000/pLE7 is an artifact of the N-Terminal yellow fluores-cent protein tag. J Bacteriol 194:6382– 6386. https://doi.org/10.1128/JB.00505-12.

8. Gan L, Chen S, Jensen GJ. 2008. Molecular organization of Gram-negative peptidoglycan. Proc Natl Acad Sci U S A 105:18953–18957.https://doi.org/10.1073/pnas.0808035105.

9. Beeby M, Gumbart JC, Roux B, Jensen GJ. 2013. Architecture and assem-bly of the Gram-positive cell wall. Mol Microbiol 88:664 – 672. https://doi.org/10.1111/mmi.12203.

10. Ebersbach G, Briegel A, Jensen GJ, Jacobs-Wagner C. 2008. A self-associating protein critical for chromosome attachment, division, andpolar organization in caulobacter. Cell 134:956 –968. https://doi.org/10.1016/j.cell.2008.07.016.

11. Iancu CV, Ding HJ, Morris DM, Dias DP, Gonzales AD, Martino A, JensenGJ. 2007. The structure of isolated Synechococcus strain WH8102 car-boxysomes as revealed by electron cryotomography. J Mol Biol 372:764 –773. https://doi.org/10.1016/j.jmb.2007.06.059.

Dobro et al. Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 12

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

12. Murphy GE, Leadbetter JR, Jensen GJ. 2006. In situ structure of thecomplete Treponema primitia flagellar motor. Nature 442:1062–1064.https://doi.org/10.1038/nature05015.

13. Chen S, Beeby M, Murphy GE, Leadbetter JR, Hendrixson DR, Briegel A,Li Z, Shi J, Tocheva EI, Muller A, Dobro MJ, Jensen GJ. 2011. Structuraldiversity of bacterial flagellar motors. EMBO J 30:2972–2981. https://doi.org/10.1038/emboj.2011.186.

14. Abrusci P, Vergara-Irigaray M, Johnson S, Beeby MD, Hendrixson DR,Roversi P, Friede ME, Deane JE, Jensen GJ, Tang CM, Lea SM. 2013.Architecture of the major component of the type III secretion systemexport apparatus. Nat Struct Mol Biol 20:99 –104.

15. Chang YW, Rettberg LA, Treuner-Lange A, Iwasa J, Sogaard-Andersen L,Jensen GJ. 2016. Architecture of the type IVa pilus machine. Science351:aad2001. https://doi.org/10.1126/science.aad2001.

16. Briegel A, Li X, Bilwes AM, Hughes KT, Jensen GJ, Crane BR. 2012.Bacterial chemoreceptor arrays are hexagonally packed trimers ofreceptor dimers networked by rings of kinase and coupling proteins.Proc Natl Acad Sci U S A 109:3766 –3771. https://doi.org/10.1073/pnas.1115719109.

17. Tocheva EI, Matson EG, Morris DM, Moussavi F, Leadbetter JR, Jensen GJ.2011. Peptidoglycan remodeling and conversion of an inner membraneinto an outer membrane during sporulation. Cell 146:799 – 812. https://doi.org/10.1016/j.cell.2011.07.029.

18. Basler M, Pilhofer M, Henderson GP, Jensen GJ, Mekalanos JJ. 2012. TypeVI secretion requires a dynamic contractile phage tail-like structure.Nature 483:182–186. https://doi.org/10.1038/nature10846.

19. Guerrero-Ferreira RC, Viollier PH, Ely B, Poindexter JS, Georgieva M,Jensen GJ, Wright ER. 2011. Alternative mechanism for bacteriophageadsorption to the motile bacterium Caulobacter crescentus. Proc NatlAcad Sci U S A 108:9963–9968. https://doi.org/10.1073/pnas.1012388108.

20. Oikonomou CM, Jensen GJ. 2016 A new view into prokaryotic cellbiology from electron cryotomography. Nat Rev Microbiol 14:205–220.https://doi.org/10.1038/nrmicro.2016.7.

21. Ingerson-Mahar M, Briegel A, Werner JN, Jensen GJ, Gitai Z. 2010. Themetabolic enzyme CTP synthase forms cytoskeletal filaments. Nat cellbiol 12:739 –746. https://doi.org/10.1038/ncb2087.

22. Briegel A, Ding HJ, Li Z, Werner J, Gitai Z, Dias DP, Jensen RB, Jensen GJ.2008. Location and architecture of the Caulobacter crescentus chemore-ceptor array. Mol Microbiol 69:30 – 41. https://doi.org/10.1111/j.1365-2958.2008.06219.x.

23. Chang YW, Chen S, Tocheva EI, Treuner-Lange A, Lobach S, Sogaard-Andersen L, Jensen GJ. 2014. Correlated cryogenic photoactivated lo-calization microscopy and cryo-electron tomography. Nat Methods 11:737–739. https://doi.org/10.1038/nmeth.2961.

24. Murphy GE, Matson EG, Leadbetter JR, Berg HC, Jensen GJ. 2008. Novelultrastructures of Treponema primitia and their implications for motility.Mol Microbiol 67:1184 –1195. https://doi.org/10.1111/j.1365-2958.2008.06120.x.

25. Muller A, Beeby M, McDowall AW, Chow J, Jensen GJ, Clemons WM Jr.2014. Ultrastructure and complex polar architecture of the humanpathogen Campylobacter jejuni. Microbiologyopen 3:702–710.

26. Ding HJ, Oikonomou CM, Jensen GJ. 2015. The caltech tomographydatabase and automatic processing pipeline. J Struct Biol 192:279 –286.https://doi.org/10.1016/j.jsb.2015.06.016.

27. Dobro MJ, Piper A. 2017. Uncharacterized bacterial structures re-vealed by electron cryotomography. figshare https://figshare.com/s/782461843c3150d27cfa. Retrieved 23 June 2017.

28. Staley JT, Bont JA, Jonge K. 1976. Prosthecobacter fusiformis nov. gen. etsp., the fusiform caulobacter. Antonie Van Leeuwenhoek 42:333–342.https://doi.org/10.1007/BF00394132.

29. Gonin M, Quardokus EM, O’Donnol D, Maddock J, Brun YV. 2000. Reg-ulation of stalk elongation by phosphate in. Caulobacter crescentus. JBacteriol 182:337–347.

30. Wagner JK, Setayeshgar S, Sharon LA, Reilly JP, Brun YV. 2006. A nutrientuptake role for bacterial cell envelope extensions. Proc Natl Acad sciU S A 103:11772–11777. https://doi.org/10.1073/pnas.0602047103.

31. Schlimpert S, Klein EA, Briegel A, Hughes V, Kahnt J, Bolte K, Maier UG,Brun YV, Jensen GJ, Gitai Z, Thanbichler M. 2012. General protein diffu-sion barriers create compartments within bacterial cells. Cell 151:1270 –1282. https://doi.org/10.1016/j.cell.2012.10.046.

32. De Troch P, Vanderleyden J. 1996. Surface Properties and motility ofrhizobium and azospirillum in relation to plant root attachment. MicrobEcol 32:149 –169. https://doi.org/10.1007/BF00185885.

33. Steenhoudt O, Vanderleyden J. 2000. Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemicaland ecological aspects. FEMS Microbiol Rev 24:487–506. https://doi.org/10.1111/j.1574-6976.2000.tb00552.x.

34. Mukherjee T, Kumar D, Burriss N, Xie Z, Alexandre G. 2016. Azospirillumbrasilense chemotaxis depends on two signaling pathways regulatingdistinct motility parameters. J Bacteriol 198:1764 –1772. https://doi.org/10.1128/JB.00020-16.

35. Bible AN, Khalsa-Moyers GK, Mukherjee T, Green CS, Mishra P, Purcell A,Aksenova A, Hurst GB, Alexandre G. 2015. Metabolic adaptations ofAzospirillum brasilense to oxygen stress by cell-to-cell clumping andflocculation. Appl Environ Microbiol 81:8346 – 8357. https://doi.org/10.1128/AEM.02782-15.

36. Bible A, Russell MH, Alexandre G. 2012. The Azospirillum brasilense Che1chemotaxis pathway controls swimming velocity, which affects transientcell-to-cell clumping. J Bacteriol 194:3343–3355. https://doi.org/10.1128/JB.00310-12.

37. Moissl C, Rachel R, Briegel A, Engelhardt H, Huber R. 2005. The uniquestructure of archaeal ‘hami’, highly complex cell appendages with nano-grappling hooks. Mol Microbiol 56:361–370. https://doi.org/10.1111/j.1365-2958.2005.04294.x.

38. Epstein EA, Reizian MA, Chapman MR. 2009. Spatial clustering of thecurlin secretion lipoprotein requires curli fiber assembly. J Bacteriol191:608 – 615. https://doi.org/10.1128/JB.01244-08.

39. Van Gerven N, Klein RD, Hultgren SJ, Remaut H. 2015. Bacterial amyloidformation: Structural insights into curli biogensis. Trends Microbiol 23:693–706. https://doi.org/10.1016/j.tim.2015.07.010.

40. Dueholm MS, Albertsen M, Otzen D, Nielsen PH. 2012. Curli functionalamyloid systems are phylogenetically widespread and display largediversity in operon and protein structure. PLoS One 7:e51274. https://doi.org/10.1371/journal.pone.0051274.

41. Cameron DE, Urbach JM, Mekalanos JJ. 2008. A defined transposonmutant library and its use in identifying motility genes in Vibrio cholerae.Proc Natl Acad Sci U S A 105:8736 – 8741. https://doi.org/10.1073/pnas.0803281105.

42. Weiner RM, Melick M, O’Neill K, Quintero E. 2000. Hyphomonas adhaerenssp. nov., Hyphomonas johnsonii sp. nov. and Hyphomonas rosenbergii sp.nov., marine budding and prosthecate bacteria. Int J Syst Evol Microbiol50 Part 2:459 – 469.

43. Zerfas PM, Kessel M, Quintero EJ, Weiner RM. 1997. Fine-structure evi-dence for cell membrane partitioning of the nucleoid and cytoplasmduring bud formation in Hyphomonas species. J Bacteriol 179:148 –156.https://doi.org/10.1128/jb.179.1.148-156.1997.

44. Pommier Y, Covey JM, Kerrigan D, Markovits J, Pham R. 1987. DNAunwinding and inhibition of mouse leukemia L1210 DNA topoisomeraseI by intercalators. Nucleic Acids Res 15:6713– 6731. https://doi.org/10.1093/nar/15.16.6713.

45. Wolf SG, Frenkiel D, Arad T, Finkel SE, Kolter R, Minsky A. 1999. DNAprotection by stress-induced biocrystallization. Nature 400:83– 85.https://doi.org/10.1038/21918.

46. Teramoto J, Yoshimura SH, Takeyasu K, Ishihama A. 2010. A novelnucleoid protein of Escherichia coli induced under anaerobiotic growthconditions. Nucleic Acids Res 38:3605–3618. https://doi.org/10.1093/nar/gkq077.

47. Lim CJ, Lee SY, Teramoto J, Ishihama A, Yan J. 2013. The nucleoid-associated protein Dan organizes chromosomal DNA through rigid nu-cleoprotein filament formation in E. coli during anoxia. Nucleic Acids Res41:746 –753. https://doi.org/10.1093/nar/gks1126.

48. Egelman EH, Stasiak A. 1986. Structure of helical RecA-DNA complexes.Complexes formed in the presence of ATP-gamma-S or ATP. J Mol Biol191:677– 697.

49. Mizuno N, Dramicanin M, Mizuuchi M, Adam J, Wang Y, Han YW, YangW, Steven AC, Mizuuchi K, Ramon-Maiques S. 2013. MuB is an AAA�ATPase that forms helical filaments to control target selection for DNAtransposition. Proc Natl Acad Sci U S A 110:E2441–E2450. https://doi.org/10.1073/pnas.1309499110.

50. Barry RM, Gitai Z. 2011. Self-assembling enzymes and the origins of thecytoskeleton. Curr Opin Microbiol 14:704 –711. https://doi.org/10.1016/j.mib.2011.09.015.

51. Matayoshi S, Oda H, Sarwar G. 1989. Relationship between the produc-tion of spirosomes and anaerobic glycolysis activity in Escherichia coli B.J Gen Microbiol 135:525–529.

52. Laurenceau R, Krasteva PV, Diallo A, Ouarti S, Duchateau M, MalosseC, Chamot-Rooke J, Fronzes R. 2015. Conserved Streptococcus pneu-

ECT of Novel Bacterial Structures Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 13

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from

moniae spirosomes suggest a single type of transformation pilus incompetence. PLoS Pathog 11:e1004835. https://doi.org/10.1371/journal.ppat.1004835.

53. Swulius MT, Chen S, Jane Ding H, Li Z, Briegel A, Pilhofer M, Tocheva EI,Lybarger SR, Johnson TL, Sandkvist M, Jensen GJ. 2011. Long helicalfilaments are not seen encircling cells in electron cryotomograms ofrod-shaped bacteria. Biochem Biophys Res Commun 407:650 – 655.https://doi.org/10.1016/j.bbrc.2011.03.062.

54. Jones LJ, Carballido-Lopez R, Errington J. 2001. Control of cell shape inbacteria: helical, actin-like filaments in Bacillus subtilis. Cell 104:913–922.https://doi.org/10.1016/S0092-8674(01)00287-2.

55. Shih YL, Le T, Rothfield L. 2003. Division site selection in Escherichia coliinvolves dynamic redistribution of Min proteins within coiled structuresthat extend between the two cell poles. Proc Natl Acad Sci U S A100:7865–7870. https://doi.org/10.1073/pnas.1232225100.

56. Scheffel A, Gruska M, Faivre D, Linaroudis A, Plitzko JM, Schuler D.2006. An acidic protein aligns magnetosomes along a filamentousstructure in magnetotactic bacteria. Nature 440:110 –114. https://doi.org/10.1038/nature04382.

57. Szwedziak P, Wang Q, Bharat TA, Tsim M, Lowe J. 2014. Architecture ofthe ring formed by the tubulin homologue FtsZ in bacterial cell division.eLife 9:e04601. doihttps://doi.org/10.7554/elife.04601.

58. Kuhn J, Briegel A, Morschel E, Kahnt J, Leser K, Wick S, Jensen GJ,Thanbichler M. 2010. Bactofilins, a ubiquitous class of cytoskeletal pro-teins mediating polar localization of a cell wall synthase in Caulobactercrescentus. EMBO J 29:327–339. https://doi.org/10.1038/emboj.2009.358.

59. Aylett CH, Wang Q, Michie KA, Amos LA, Lowe J. 2010. Filament structureof bacterial tubulin homologue TubZ. Proc Natl Acad Sci U S A 107:19766 –19771. https://doi.org/10.1073/pnas.1010176107.

60. Bharat TA, Murshudov GN, Sachse C, Lowe J. 2015. Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.Nature 523:106 –110. https://doi.org/10.1038/nature14356.

61. Santarella-Mellwig R, Franke J, Jaedicke A, Gorjanacz M, Bauer U, Budd A,Mattaj IW, Devos DP. 2010. The compartmentalized bacteria of thePlanctomycetes-Verrucomicrobia-Chlamydiae superphylum have mem-brane coat-like proteins. PLoS biology 8:e1000281. https://doi.org/10.1371/journal.pbio.1000281.

62. Lonhienne TG, Sagulenko E, Webb RI, Lee KC, Franke J, Devos DP,Nouwens A, Carroll BJ, Fuerst JA. 2010. Endocytosis-like protein uptakein the bacterium Gemmata obscuriglobus. Proc Natl Acad Sci U S A107:12883–12888. https://doi.org/10.1073/pnas.1001085107.

63. Makarova KS, Koonin EV. 2010. Two new families of the FtsZ-tubulinprotein superfamily implicated in membrane remodeling in diversebacteria and archaea. Biol Direct 5:33. https://doi.org/10.1186/1745-6150-5-33.

64. Briegel A, Ortega DR, Tocheva EI, Wuichet K, Li Z, Chen S, Müller A, Iancu

CV, Murphy GE, Dobro MJ, Zhulin IB, Jensen GJ. 2009. Universal archi-tecture of bacterial chemoreceptor arrays. Proc Natl Acad Sci U S A106:17181–17186. https://doi.org/10.1073/pnas.0905181106.

65. Briegel A, Ortega DR, Mann P, Kjær A, Ringgaard S, Jensen GJ. 2016.Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibriocholerae and are stabilized by a double signaling domain receptorDosM. Proc Natl Acad Sci U S A 113:10412–10417. https://doi.org/10.1073/pnas.1604693113.

66. Briegel A, Ames P, Gumbart JC, Oikonomou CM, Parkinson JS, Jensen GJ.2013. The mobility of two kinase domains in the Escherichia coli chemo-receptor array varies with signalling state. Mol Microbiol 89:831– 841.https://doi.org/10.1111/mmi.12309.

67. Briegel A, Beeby M, Thanbichler M, Jensen GJ. 2011. Activated chemo-receptor arrays remain intact and hexagonally packed. Mol Microbiol82:748 –757. https://doi.org/10.1111/j.1365-2958.2011.07854.x.

68. Cserti E, Rosskopf S, Chang YW, Eisheuer S, Selter L, Shi J, Regh C, KoertU, Jensen GJ, Thanbichler M. 2017. Dynamics of the peptidoglycanbiosynthetic machinery in the stalked budding bacterium Hyphomonasneptunium. Mol Microbiol 103:875– 895. https://doi.org/10.1111/mmi.13593.

69. Tivol WF, Briegel A, Jensen GJ. 2008. An improved cryogen for plungefreezing. Microsc Microanal 14:375–379. https://doi.org/10.1017/S1431927608080781.

70. Suloway C, Shi J, Cheng A, Pulokas J, Carragher B, Potter CS, Zheng SQ,Agard DA, Jensen GJ. 2009. Fully automated, sequential tilt-series acqui-sition with Leginon. J Struct Biol 167:11–18. https://doi.org/10.1016/j.jsb.2009.03.019.

71. Zheng SQ, Keszthelyi B, Branlund E, Lyle JM, Braunfeld MB, Sedat JW,Agard DA. 2007. UCSF tomography: an integrated software suite forreal-time electron microscopic tomographic data collection, alignment,and reconstruction. J Struct Biol 157:138 –147. https://doi.org/10.1016/j.jsb.2006.06.005.

72. Kremer JR, Mastronarde DN, McIntosh JR. 1996. Computer visualizationof three-dimensional image data using IMOD. J Struct Biol 116:71–76.https://doi.org/10.1006/jsbi.1996.0013.

73. Amat F, Moussavi F, Comolli LR, Elidan G, Downing KH, Horowitz M.2008. Markov random field based automatic image alignment for elec-tron tomography. J Struct Biol 161:260 –275. https://doi.org/10.1016/j.jsb.2007.07.007.

74. Nicastro D, Schwartz C, Pierson J, Gaudette R, Porter ME, McIntosh JR.2006. The molecular architecture of axonemes revealed by cryoelectrontomography. Science 313:944 –948. https://doi.org/10.1126/science.1128618.

75. Piper AT. 2017. Cell visualization VR. https://play.google.com/store/apps/details?id�com.BishopVisual.Mk2&rdid�com.BishopVisual.Mk2. Retrieved 23 June 2017.

Dobro et al. Journal of Bacteriology

September 2017 Volume 199 Issue 17 e00100-17 jb.asm.org 14

on Septem

ber 7, 2017 by CA

LIFO

RN

IA IN

ST

ITU

TE

OF

TE

CH

NO

LOG

Yhttp://jb.asm

.org/D

ownloaded from